Table 1.
List of primers that were used for the tissue distribution in C. sapidus.
Table 2.
List of primers that were used for the qPCR assays.
Fig 1.
BUSCO assessment results of the Trinity and Trinity_95 transcriptome against the Arthropoda reference.
Table 3.
Summary of Illumina sequencing statistics and mapping.
Fig 2.
UpSet plot depicting the number of unique and shared transcripts with TPM ≥ 1 in each tissue.
Intersection size represents the number of transcripts for each designated sets or groups. The dots and lines illustrate the groups defined as: HP: Hepatopancreas, YO at IM: Y-organ at intermolt, YO at iePM: Y-organ at induced early premolt and EG: Eyestalk ganglia.
Table 4.
Abundance of genes in transcript per million (TPM) and their corresponding fragments per kilo base per million mapped reads (FPKM) involved in ecdysteroid synthesis, cholesterol uptake and transport in the YO transcriptomes of C. sapidus at intermolt (IM) and induced early premolt (iePM).
When a gene presented several transcripts, the abundance of both transcripts is shown.
Table 5.
List of the candidate genes and their associated conserved domains.
Fig 3.
Phylogenic trees were generated using the default settings of Phylogeney.fr (http://www.phylogeny.fr/simple_phylogeny.cgi).
Sequences were aligned using MUSCLE, trimmed with GBLOCKS, and trees were built with PhyML. The sequencing data is included in fasta format as supplemental files (S1–S5 Files). The C. sapidus sequences are noted with an arrow in A. Neverland (Accession No MW556747);B. Spook (Accession No MW556746);C. Apolipoprotein D-like (Accession No MW556748);D. Hormone receptor 4 (Accession No MW556749);and E. Probable cytochrome p450 49a1 (Accession No MW556750). Scale bars present fixed mutations per amino acid position. And, bootstrap values (%) supporting nodes are noted with numbers.
Fig 4.
Spatial distribution of five candidate genes (Spook, Neverland, Hormone receptor 4, probable cytochrome p450 49a1 and Apolipoprotein D-like) in the tissues of an adult female blue crab.
Each cDNA tissue sample containing 12.5 ng total RNA equivalent was amplified by PCR. The PCR products were analyzed on 1.5% agarose gel and stained with ethidium bromide. The tissues are noted as: 1 = eyestalk, 2 = thoracic ganglia complex, 3 = brain, 4 = pericardial organ, 5* = Y-organ, 6 = mandibular organ, 7 = hepatopancreas, 8 = foregut, 9 = hindgut, 10 = antennal gland, 11 = gill, 12 = abdominal muscle, 13 = hypodermis, 14 = ovary, 15 = spermatheca, 16 = heart, 17 = hemocytes. Further expression analyses were carried out using the YO cDNAs (5*).
Fig 5.
Levels of hemolymph Ecd and cholesterol at different molt stages.
The group of animals undergoing natural molt cycle are colored in light grey (Intact) while the group of eyestalk-ablated animals are colored in dark grey (Ablated). (A) Ecdysteroid concentrations (ng/ml) in the hemolymph from intact prepubertal females at intermolt (IM), early premolt (ePM) and intermediate premolt (PM) and the corresponding stages from eyestalk-ablated adult females (D7 after ablation). (B) Cholesterol (free and cholesteryl esters) concentrations (μg/ml) from intact prepubertal females at intermolt (IM), early premolt (ePM) and premolt (PM) and the corresponding stages from eyestalk-ablated adult females (D7 after ablation). The levels of both Ecd and cholesterol were not statistically different at IM and ePM between natural and ablated animals (P>0.05, Students’ t-test). Statistical difference between IM, ePM and PM in a natural molt stage was evaluated using non-parametric Kruskall-wallis and Dunn test post-hoc (P<0.05). The dots represented outliers in the dataset.
Fig 6.
Boxplot representation of the candidate gene expressions in the YO of non-ablated animals (IM) and eyestalk ablated C. sapidus (iePM).
Gene expression was measured by absolute qPCR assays and presented as the number of transcripts/μg total RNA. Non-ablated control animals are shown in light grey, eyestalk-ablated animals (ablated) in dark grey. (A) Expression of Spook. (B) Expression of Hormone receptor 4 (Hr4) and probable cytochrome p450 49a1 (cyp49a1). (C) Expression of Neverland and Apolipoprotein D-like (ApoD). The expression levels of each gene measured in non-ablated and ablated samples were examined for statistical significance by Student’s t-test. Statistical differences were noted as”*” P<0.05, “**” P<0.01. The dots represented outliers in the dataset.
Fig 7.
Boxplot representation of the candidate gene expressions in the YO of intact C. sapidus at different molt stages.
Gene expression was measured by absolute qPCR assays and expressed in the number of transcripts/μg total RNA. Molt stages are colored as followed: IM (dark pink), ePM (turquoise), and PM (dark blue). (A) Expression of Spook and probable cytochrome p450 49a1(cyp49a1). (B) Expression of Hormone receptor 4 (Hr4), Neverland, and Apoprotein D-like (ApoD). The expression levels measured at the three molt stages were analyzed using a non-parametric one-way ANOVA Kruskal-Wallis followed by a Dunn post hoc test. Statistical differences were noted as”*” P<0.05. The dots represented outliers in the dataset.
Fig 8.
Summary of the ecdysteroidogenesis pathway in C. sapidus based on the data presented in this study.
First, cholesterol concentration increased at early premolt (ePM, turquoise in Fig 7), resulting in the initial rise in ecdysone synthesis and release via upregulation of Neverland (Nvd) and Spook. Then this initially elevated ecdysone in hemolymph upregulated Apolipoprotein D-like (ApoD) at premolt (PM, dark blue, in Fig 7) for further increase in cholesterol transport for ecdysteroid synthesis. It also upregulated other ecdysone responsive genes, including probable cytochrome p450 49a1 (cyp49a1) and Hormone receptor 4 (HR4) at PM. The dashed line represents the rest of the ecdysteroidogenesis steps not included in this study. The upward arrows illustrate the upregulation. 7dC: 7-dehydrocholesterol.